U.S. Pat. No. 6,594,011 issued Jul. 15, 2003, the entirety of which is incorporated by reference herein for all purposes, discloses an imaging apparatus and method for real time imaging ellipsometry for high throughput sensing of binding events useful in molecular interaction analysis including biotech applications. The apparatus disclosed employs the immobilization of an array of binding or capture molecules (“ligands”) on a planar surface of a transparent substrate and the use of a beam of polarized light directed at the underside of the surface in a manner to achieve total internal reflection (TIR) and generate an evanescent field in the plane of the ligands. The ligands are exposed to a biological sample and analytes in the biological sample bind to different patterns of the immobilized ligands in a manner to change the polarization at locations in the array at which binding occurs. An image of the array is compared with a stored image of the initial light polarization shifts to determine the location and magnitude of binding events within the array, thus identifying and quantitating the analytes present in the biological sample.
The apparatus for implementing the foregoing technique typically employs a prism or gratings to achieve the requisite TIR generated evanescent field, the prism being the most practical implementation.
TIR imaging ellipsometry works well for fields of view up to 1-2 cm2, which permits real time imaging of tens of thousands of binding events simultaneously. However, there is a need to be able to image or scan areas which are much larger, such as 128 mm×86 mm (e.g. the area of a 384 well or a 96 well multiwell plate), to permit lower costs per test and for multiple tests per patient for large numbers of patients simultaneously, which is increasingly a requirement for clinical diagnostics and personalized medicine. Obviating the need for a prism simplifies both the instrument and disposable multiwell plate.
Also, it is well known in the art to label a hybridized target or probe by, for example, adding a molecule, either being conjugated to, bound to, or associated with the target. Labels include reporter molecules that can be detected directly by virtue of generating a signal. Examples include but are not limited to fluorophores, dyes, chemiluminescent probes, radioactive atoms or molecules, magnetic particles and quantum dots. The detection of labeled target molecules in a binding assay may employ total internal reflection as a way of exciting fluorophores, for example, which could then be detected from above or below the assay surface. There are examples in the literature which demonstrate the benefits of detecting fluorophore emission at angles close to the critical angle (see e.g., http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfintro.html) in such a total internal reflection configuration. The reason for this has to do with the anisotropic fluorescence emission intensity at a reflection interface between higher and lower index materials. In these other labeled examples, the light need not be polarized.